Action spectroscopy of the isolated red Kaede fluorescent protein chromophore

Incorporation of fluorescent proteins into biochemical systems has revolutionized the field of bioimaging. In a bottom-up approach, understanding the photophysics of fluorescent proteins requires detailed investigations of the light-absorbing chromophore, which can be achieved by studying the chromophore in isolation. This paper reports a photodissociation action spectroscopy study on the deprotonated anion of the red Kaede fluorescent protein chromophore, demonstrating that at least three isomers-assigned to deprotomers-are generated in the gas phase. Deprotomer-selected action spectra are recorded over the S-1 & LARR; S-0 band using an instrument with differential mobility spectrometry coupled with photodissociation spectroscopy. The spectrum for the principal phenoxide deprotomer spans the 480-660 nm range with a maximum response at & AP;610 nm. The imidazolate deprotomer has a blue-shifted action spectrum with a maximum response at & AP;545 nm. The action spectra are consistent with excited state coupled-cluster calculations of excitation wavelengths for the deprotomers. A third gas-phase species with a distinct action spectrum is tentatively assigned to an imidazole tautomer of the principal phenoxide deprotomer. This study highlights the need for isomer-selective methods when studying the photophysics of biochromophores possessing several deprotonation sites.

Automated summary

Incorporation of fluorescent proteins into biochemical systems has revolutionized bioimaging. A study on the red Kaede fluorescent protein chromophore in the gas phase revealed three isomers, highlighting the need for isomer-selective methods in studying biochromophores.